Aberrant bone formation, a primary factor in osteoarthritis (OA) joint pathology, is a key issue when proposing treatments for the clinical symptoms associated with OA. In rheumatoid arthritis (RA) research, inhibition of Dkk-1, an antagonist of the canonical Wnt signaling pathway, reversed the bone destructive effect in a transgenic mouse model of RA, but induced the formation of osteophytes, or bone spurs, a marker of OA (8). Whereas RA leads to bone resorption, OA leads to formation of new bone. One strategy to reverse the bone- forming pathology of OA may be to reverse the strategy mentioned above, an up-regulation of the Wnt- antagonist Dkk-1. Our long-term goal is to utilize a gene therapy-based approach to assess the regulatory role of Wnt signaling in the pathogenesis of OA. The specific aims of this proposal are designed to contribute to this assessment by up- and down-regulating Wnt signaling through introduction of a Wnt transgene (Wnt10b) and a Wnt-inhibiting transgene (Dkk-1). Aim 1 is focused on determining the most efficient gene transfer strategy for effective regulatory control of Wnt signaling in articular tissues. Aim 2 is focused on manipulating Wnt signaling in vivo in a rat model of OA. An improved understanding of the role of Wnt signaling in postnatal joint biology and joint remodeling in chronic arthritis will lead to improved characterization of the causes of OA pathologies and facilitate more effective treatments for this disease in the future. To achieve our goals, we intend to use an in vitro study to refine the ability of several candidate recombinant adeno-associated viral vector (rAAV) serotypes to transduce cell monolayers and tissue explants from articular joints. Cell and tissue transduction efficiency will be assessed using reporter gene assays. Cells and explants will then be transduced with Wnt10b and Dkk-1-containing vectors and gene expression and protein production will be assessed using RT-PCR and Western blotting. Fidelity of Wnt/2-catenin signaling will be assessed by Western blotting with anti-2-catenin antibodies. To determine the effectiveness of manipulating Wnt10b and Dkk-1 signaling in vivo, we will use an established rat OA model consisting of anterior cruciate ligament transection (ACLT) in combination with resection of the medial menisci (MMx), which mimics the pathogenesis of human OA in terms of cartilage degradation and osteophyte formation. Vectors containing Wnt10b and Dkk-1 transgenes will be injected into surgical and control joints and the efficacy of gene therapy-induced changes will be assessed using live-imaging and confirmatory tissue sections for digital imaging, semi-quantitative histopathological grading and Western blotting. PUBLIC HEALTH RELEVANCE: The goal of this proposed research is to identify mechanisms that regulate disease progression in osteoarthritic joints. Approximately 80% of adults show some radiographic evidence of osteoarthritis, a major contributor to functional impairment and reduced independence, after the age of age of 65. This study may lead to a less invasive and less traumatic, gene therapy-based approach to treatment of this debilitating disease.